pediatric hematology, methods and protocols

Molecular Diagnosis of FA and DC 11FANCC Exon 5 Forward, CTG ATG TAA TCC TGT TTG CAG CGT G 186 Reverse, TCC TCT CAT AAC CAA ACT GAT ACAExon 6 Forward, GTC CTT AAT TAT GCA TGG CTC TTA G 2

Molecular Diagnosis of FA and DC 3

3

From: Methods in Molecular Medicine, Vol 91: Pediatric Hematology: Methods and Protocols Edited by: N J Goulden and C G Steward © Humana Press Inc., Totowa, NJ

1

Molecular Diagnosis of Fanconi Anemia

and Dyskeratosis Congenita

Alex J Tipping, Tom J Vulliamy, Neil V Morgan, and Inderjeet Dokal

Fanconi anemia (FA) is an autosomal recessive disorder in which sive BM failure occurs in the majority of patients and in which there is anincreased predisposition to malignancy, particularly acute myeloid leukemia.Although many FA patients will have associated somatic abnormalities, approx30% will not This makes diagnosis based on clinical criteria alone difficultand unreliable FA cells characteristically show an abnormally high frequency

progres-of spontaneous chromosomal breakage and hypersensitivity to the clastogeniceffect of DNA crosslinking agents such as diepoxybutane (DEB) and mitomy-cin C (MMC) This property of the FA cell has been exploited in the “DEB/MMC stress test” for FA and has been critical in defining the FA complemen-tation groups/subtypes (FA-A, FA-B, FA-C, FA-D1, FA-D2, FA-E, FA-F, and

FA-G) and in identification of the FA genes (FANCA, FANCC, FANCD2,

FANCE, FANCF, FANCG) (3–9) The DEB/MMC test remains the front-line

diagnostic test for FA However, the DEB/MMC test is not able to distinguish

FA carriers from normals, antenatal diagnoses based on this are possible onlylater in pregnancy, and it is unable to classify a patient into an FA subgroup(complementation subtype) Because of these limitations there are circum-stances when a molecular diagnosis is desirable Furthermore molecular analy-

of prognostic significance are emerging As can be seen from Table 1, the six

FA genes identified to date collectively represent >90% of FA patients, withFA-A subtype accounting for approx 70% of FA patients However, severaldifferent mutations have been identified in each different FA gene, with more

than 100 mutations in the FANCA gene alone This means that molecular

diag-nosis for FA is very complex

Given the number of genes mutated in FA, the choice of which gene to beginscreening for mutations is obviously critical In the absence of any informationfrom techniques such as cell fusion or retroviral transduction experiments, orgeographical clustering of a particular complementation group, statistically

there is a approx 70% chance that the patient carries mutations in FANCA For

this reason we present a quantitative fluorescent multiplex genomic polymerasechain reaction (PCR) technique that was shown to detect a high frequency of

FANCA mutations in a previous study (10) Another technique (solid-phase

fluorescent chemical cleavage of mismatch [FCCM]) formed the balance of

our FANCA screening, but lack of space prevents its detailed description here The multiplex PCR technique detects but does not delimit deletions in FANCA,

which account for a high proportion (40%) of mutations in FA-A patients whoare largely compound heterozygotes Small deletions of less than a whole exon

or point mutations were detected with FCCM from reverse transcriptase-PCR(RT-PCR) generated products Consanguinity in the kindred (and hence pre-

Molecular Diagnosis of FA and DC 5dicted homozygosity) suggests caution when using single techniques for

FANCA mutation screening, owing to the risk of missing mutations of one type

or the other Used together, we found that the two techniques missed only 17%

of FANCA mutations.

The multiplex PCR technique is adaptable for other genes in which tions are present in either a homozygous or heterozygous state, with the simpleselection of primer sets that amplify exons known to be deleted in the pathol-

dele-ogy of the disease For FANCA screening we utilized the fifth and sixth exons

of FANCC, not known to be deleted in FA-C patients (11–12), or alternatively

exon 1 of myelin protein zero Use of genomic DNA in short PCRs allowscomparison of the intensity of fluorescence contributed by each exon relative

to a known diploid exon, as the reactions are still stoichiometric in the early

(pre-plateau) phase of the PCR (13) Fluorescence intensity measurement and

size discrimination (for small deletions within an exon) are achieved by the use

of fluorescently labeled primers and an ABI 373 DNA sequencer

Dyskeratosis congenita (DC) is an inherited disorder characterized by thetriad of abnormal skin pigmentation, nail dystrophy, and mucosal leucoplakia.Since its first description by Zinsser in 1906 it has become recognized that, as

in FA, the clinical phenotype is highly variable, with a variety of noncutaneous(dental, gastrointestinal, genitourinary, neurological, ophthalmic, pulmonary,and skeletal) abnormalities having been observed X-linked recessive, autoso-mal dominant, and autosomal recessive forms of the disease are recognized Inthe DC registry at the Hammersmith Hospital there are 154 families (compris-ing 199 males and 56 females) from 33 countries The clinical phenotype ishighly variable both in the age at onset and severity of a particular abnormalityand in the combination of such abnormalities in a given patient This makesdiagnosis based on clinical criteria alone difficult and unreliable particularlywhere non-cutaneous abnormalities (such as hematological abnormalities) pre-cede the classical diagnostic features A laboratory diagnostic test was there-fore very desirable Unlike the situation for FA, there is no reliable functional

phenotypic test for DC However, the identification of the DKC1 gene (14) (which is mutated in X-linked DC) and the hTR gene (15) (mutated in autoso-

mal dominant DC) now makes it possible to undertake molecular analysis in alarge subset of DC families The data from the DCR shows that approx 40–

50% of the DC patients have mutations in DKC1, and approx <10% of the

families have mutations in hTR (Table 2) This means that for the present it is

not possible to substantiate a molecular diagnosis in approx 40–50% of DCpatients and highlights the need to identify other DC causing genes As for FA,once a mutation has been identified, as well as confirming the diagnosis in the

6 Tipping et al.

Table 2

DC Subtypes

DC subtype Percentage Chromosome RNA/protein Mutations

incidencea location product identifiedX-linked recessive 40–50 Xq28 Dyskerin >25Autosomal dominant <10 3q21–3q28 hTR 5Autosomal recessive 40–50 ? ? ?

aThe approximate percentage incidences of the different subtypes are based on the Dyskeratosis Congenita Registry (DCR) at the Hammersmith Hospital.

patient, it is possible to offer carrier detection and antenatal diagnosis in at riskfamilies

The DKC1 mutations are almost always missense mutations, and the

pre-ferred strategy for their identification has been single-strand conformational

polymorphism (SSCP) analysis (16) The procedure is detailed in this chapter.

More recently we have been screening for point mutation on the TransgenomicWave DNA fragment analysis system In this procedure, the patient PCR prod-ucts are mixed with equivalent wild-type products, denatured, and cooledslowly to allow for possible heteroduplex formation and then analysed byreverse-phase ion-pair high-performance liquid chromatography (HPLC).When this procedure is carried out at a partially denaturing temperature,heteroduplex DNA elutes from the column earlier in a gradient of acetonitrilethan the fully base-paired homoduplex DNA Peaks of DNA elution arerecorded and disturbances to the highly reproducible normal pattern obtainedare indicative of the presence of mutation This method is simple in that it doesnot require the use of radiolabeling and gel electrophoresis and may become amore widespread as the equipment becomes available

2 Materials

2.1 Genomic DNA Purification

1 1X SET buffer: 10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 1 mM EDTA.

2 10% (w/v) sodium dodecyl sulfate (SDS)

3 10 mg/mL of proteinase K in water

4 6 M NaCl.

5 Isoamyl alcohol:chloroform 1:24

6 Cold (–20°C) absolute ethanol

7 TE: 10 mM Tris-HCl, pH 7.5, 1 mM EDTA.

Molecular Diagnosis of FA and DC 7

2.2 PCR

1 Taq polymerase and oligonucleotide primers: These can be purchased from a

variety of different companies The oligos are usually 18–22 bases in length Forthe FA multiplex PCRs the forward primer for each exon must be fluorescentlylabeled

2 PCR buffers: These are usually supplied along with the Taq polymerase For FA multiplex PCR the buffer composition is 67 mM Tris-HCl, pH 8.8, 16.6 mM

(NH4)2SO4, 1.5 mM MgCl2, 0.17 mg/mL of bovine serum albumin (BSA) For

the DKC1 and hTR genes the 10X buffer (from Advanced Biotechnologies) is:

750 mM Tris-HCl, pH 8.8, 200 mM (NH4)2SO4, 0.1% (v/v) Tween 20 A solution

of 25 mM MgCl2 is also provided and added separately to the PCR reaction

3 2 mM and 10 mM dNTP.

4 Dimethyl sulfoxide (DMSO)

2.3 Multiplex Electrophoresis and Fluorescent Detection

1 5% Denaturing polyacrylamide gel (poured according to recommendations foruse with ABI Genescan)

2 10X TBE running buffer (see Subheading 2.4.).

3 Formamide loading buffer: 95% formamide in 1X TBE with 5 mg/mL of tran blue

dex-4 Internal size standard: Genescan-500 ROX (PE Biosystems)

2 A slab gel dryer with vacuum pump

3 10X Tris borate EDTA (TBE) buffer : Add 216 g of Trizma base, 18.6 g of EDTA,and 110 g of orthoboric acid to 1600 mL of water, dissolve and top up to 2 L;dilute 1:10 for use as 1X TBE buffer

4 Routine SSCP gel mix: For an 80-mL gel, take 53.6 mL of H2O, 8 mL 10X TBE,

4 mL of glycerol, 12 mL 40% (w/v) acrylamide solution (Caution: acrylamide is

a potent neurotoxin), and 2.4 mL of 2% (w/v) bis-acrylamide solution.

5 10% (w/v) ammonium persulfate This reagent is not stable at room temperature

It can be kept for only a few weeks on the bench, and should be stored at –20°C

6 TEMED: N,N,N',N'-Tetramethylethylenediamine.

7 Formamide dye: to 10 mL of deionized formamide, add 10 mg of xylene cyanol

FF, 10 mg of bromophenol blue, and 200 µL of 0.5 mol/L of EDTA

8 Tipping et al.

3 Methods

3.1 DNA Preparation

Prepare genomic DNA from lymphoblastoid cell lines (see Note 1) by salt–

chloroform extraction,essentially as described elsewhere (17): In brief:

1 Resuspend the cell pellet in 4.5 mL of 1X SET

2 Add 250 µL of 10% SDS and 100 µL of 10 mg/mL of proteinase K, mix, andleave at 37°C overnight

3 If clear, proceed If not, add a further 100 µL of proteinase K and continue bation for 2–3 h

incu-4 Add prewarmed (37°C) 6 M NaCl to a final concentration of 1.5 M (i.e., for 4.5 mL, add 1.5 mL 6 M of NaCl).

5 Add an equal volume of isoamyl alcohol–chloroform, and place on a rolling mixerfor 30–60 min

6 Centrifuge at 2000 rpm for 10 min at room temperature

7 Remove the upper aqueous layer and add two volumes of cold absolute ethanol.Mix by inversion two or three times Place at –20°C for 1 h or longer

8 Centrifuge at 2000 rpm for 10 min at 4°C Remove the supernatant and wash thepellet twice with 70% ethanol

9 Briefly air-dry pellet and resuspend in TE (see Note 2).

3.2 Fluorescent Multiplex PCR for the FANCA Gene

1 IncubateDNAsamples at 55°C for 1h to redissolve fully the DNA (allowingaccurate measurement of DNA concentration) Take an aliquot of thissample todetermine the concentration by A260 measurement, anddilutethe remainder in

1X Taq DNA polymerasebuffer, and a 200 µMconcentration of each dNTP Ofeachof the primer pairs,0.2 µM worked wellfor each of themultiplexes, apart from0.4 µM for FANCAexons 5, 11, 12,and 31

After aninitial denaturation at 94°Cfor 3 min, “hot-start” the reaction withthe addition of 1.5U of Taq DNA polymerase(Promega), and perform 18 PCRcycles of: 93°C for1 min, annealing for1 min at either60°C (multiplexes 1, 3,and4) or 58°C (multiplex2), and extension for2 min at 72°C, followed byafinal extension for5 min at 72°C at the end of the 18 cycles

Molecular Diagnosis of FA and DC 9

3.3 ABI Gel Electrophoresis and Data Analysis

1 Add an aliquot ofthe PCR product (4µL) to3.5 mL of formamide loadingbuffer(95% formamide in1X TBE and5 mg of dextran blue/mL)and 0.5 of mL internallane size standard (Genescan-500Rox; PE Biosystems)

2 Denature thesamples for 5 min at 94°Cand electrophorese on a5% denaturingpolyacrylamide gelat 45 W for6 h on anABI 373 fluorescent DNAsequencer(according to the manufacturer’s instructions, omitted here for economy ofspace) Up to 24 samples can be run on each gel, remembering to include fourcontrol DNAs known to be undeleted in any of the exons under test

3 Data are analyzed by means ofGenescan and Genotyper software,to obtain trophoretograms foreach sample The positionof the peaks indicatesthe size (inbasepairs)of the exons amplified,and the areas underthe peaks indicate theamount of fluorescence fromthe product

elec-4 Thecopy number of eachexon amplified is establishedby importing the peakarea values into anExcel spreadsheet and calculatinga dosage quotient foreachexon relative toall the other amplifiedexons in patients andcontrols (for anexamplesee Table 4).

Choose peak areasfrom the best two control samples(with approximatelyequal values), and calculatedosage quotient values from them as below; valuesaretypically within the range0.77–1.25 (13) (see Note 3).

Essentially the calculation takes the average peakarea of an exon from thesecontrolsand compares this with the peak areaof the same exon fromthe patientsamples Asan example (see Table 4), in patient X the dosage quotientfor

FANCA exon 10 and FANCC exon 5is given by DQFANCA exon 10/FANCC

exon5 and is calculatedby:

[sample FANCA exon 10 peak area/sample FANCCexon 5 peak area] /[control

FANCA exon 10 peak area/control FANCC exon5 peak area] =

[1857/5301]/[6034/8180] = 0.47

The threshold for classification as heterozygous for an exon of interest isgenerally a DQ < 0.77 Good quality data are often significantly closer to 0.5

for heterozygously deleted, and 1.0 for homozygously intact (see Table 4 for

sample data) Clearly, homozygous deletions are easily confirmed by tional PCR

conven-The results for each patient are generally collated and examined cally to determine the next step of the investigation It is also wise to take anoverview of detection rates for each multiplex to determine whether a simpler,achievable multiplex reaction could expedite rapid screening of a large num-

periodi-ber of samples (see Notes 4 and 5).

10 Forward, GAT TGT AGA AGT CTT GAT GGA TGT G 259

Reverse, ATT TGG CAG ACA CCT CCC TGC TGC

11 Forward, GAT GAG CCT GAG CCA CAG TTT GTG 301

Reverse, AGA ATT CCT GGC ATC TCC AGT CAG

12 Forward, CCA CAA CTT TTT GAT CTC TGA CTT G 224

Reverse, GTG CCG TCC ACG GCA GGC AGC ATG

31 Forward, CAC ACT GTC AGA GAA GCA CAG CCA 205

Reverse, CAC GCG GCT TAA ATG AAG TGA ATG C

32 Forward, CTT GCC CTG TCC ACT GTG GAG TCC 369

Reverse, CTC ACT ACA AAG AAC CTC TAG GAC

FANCC

Exon 5 Forward, CTG ATG TAA TCC TGT TTG CAG CGT G 186

Reverse, TCC TCT CAT AAC CAA ACT GAT ACAExon 6 Forward, GTC CTT AAT TAT GCA TGG CTC TTA G 293

Reverse, CCA ACA CAC CAC AGC CTT CTA AG

Multiplex 2

FANCA

Exon 5 Forward, ACC TGC CCG TTG TTA CTT TTA 250

Reverse, AGA ACA TTG CCT GGA ACA CTGExon 17 Forward, CCC TCC ATG CCC ACT CCT CAC ACC 207

Reverse, AAA AGA AAC TGG ACC TTT GCA TExon 35 Forward, GAT CCT CCT GTC AGC TTC CTG TGA G 315

Reverse, GCA TTT TCC CTG AGA TGG TAA CAC CExon 43 Forward, GCC TGG CTG GCA ATA CAA CTC GAC 223

Reverse, GGC AGG TCC CGT CAG AAG AGA TGA G

Molecular Diagnosis of FA and DC 11

FANCC

Exon 5 Forward, CTG ATG TAA TCC TGT TTG CAG CGT G 186

Reverse, TCC TCT CAT AAC CAA ACT GAT ACAExon 6 Forward, GTC CTT AAT TAT GCA TGG CTC TTA G 293

Reverse, CCA ACA CAC CAC AGC CTT CTA AG

Multiplex 3

FANCA

Exon 21 Forward, CAG GCT CAT ACT GTA CAC AG 335

Reverse, CAC CGG CTT GAG CTG GCA CAGExon 27 Forward, CAG GCC ATC CAG TTC GGA ATG 285

Reverse, CCT TCC GGT CCG AAA GCT GC

FANCC

Exon 5 Forward, CTG ATG TAA TCC TGT TTG CAG CGT G 186

Reverse, TCC TCT CAT AAC CAA ACT GAT ACAExon 6 Forward, GTC CTT AAT TAT GCA TGG CTC TTA G 293

Reverse, CCA ACA CAC CAC AGC CTT CTA AG

Multiplex 4

FANCA

Exon 5 Forward, ACC TGC CCG TTG TTA CTT TTA 250

Reverse, AGA ACA TTG CCT GGA ACA CTGExon 11 Forward, GAT GAG CCT GAG CCA CAG TTT GTG 301

Reverse, AGA ATT CCT GGC ATC TCC AGT CAGExon 17 Forward, CCC TCC ATG CCC ACT CCT CAC ACC 207

Reverse, AAA AGA AAC TGG ACC TTT GCA TExon 21 Forward, CAG GCT CAT ACT GTA CAC AG 335

Reverse, CAC CGG CTT GAG CTG GCA CAGExon 31 Forward, CAC ACT GTC AGA GAA GCA CAG CCA 308

Reverse, CCC AAA GTT CTG GGA TTA CAG GCG TGMyelin protein zero

Exon 1 Forward, CAG TGG ACA CAA AGC CCT CTG TGT A 389

Reverse, GAC ACC TGA GTC CCA AGA CTC CCA G

12 Tipping et al.

Table 4

Statistical Profile of FANCA Dosage Multiplex 1

Peak area in Dosage Quotient inExon Control Patient Xa FANCC FANCC

aHeterozygous for a deletion of exons 10–12.

3.4 PCR Amplification of the DKC1 and hTR Genes

Oligonucleotide sequences and annealing temperatures for the PCR

ampli-fication of the 15 exons of the DKC1 gene and the hTR gene are given in

Table 5 The standard composition of the PCR mix for varying numbers of

25 µL reactions are given in Table 6 This composition works for all primers

in Table 5 except for the hTR reaction, to which 10% DMSO must be added

and the volume of H2O reduced accordingly Cycling conditions used are 95°Cfor 5 min, followed by 30 cycles of 58°C for 45 s, 72°C for 1 min, and 94°C for

45 s, followed by 58°C for 45 s and final extension at 72°C for 5 min

Restriction enzyme digestion of the PCR products can be performed as

detailed in Table 5 These are performed overnight at the appropriate

tempera-ture using the buffers supplied with the enzymes

3.5 SSCP Gel Electrophoresis

The following procedure describes the preparation of a large, thin (34 cm ×

40 × 0.4 mm) 6% nondenaturing polyacrylamide gel

1 Clean the glass plates thoroughly with detergent and a scourer Rinse well anddry Swab the larger plate with 100% ethanol Treat one surface of the smallerplate with a siliconizing solution or a nontoxic gel coating solution (e.g., GelSlick from FMC), by applying a small amount, a few milliliters, and buffing drywith a paper towel Assemble the gel using spacers, bulldog clips, and electrical

Molecular Diagnosis of FA and DC 13

Table 5

Primer Pairs used in the Amplification of the DKC1 Exons and the hTR Gene

RE Cut Oligo Sequence Exon Temperature Size enzyme sizes(bp) PH7 CCGAGCCAGCAAATCGCATT 1 60 318 StyI 189, 145 Ex1R CGGGAACCAGAGGGAGGCGTG

AAF1 AATCCATTTCCTACCTGCCC 2 60 159

AAR1 CAATGCTGGCCCATTCCTTG

BBF1 AAAGGCATACATTTCCATGG 3 58 268 HinfI 147, 121 BBR1 CAAGGATGCCAGCAGTAAG

CCF1 GCCACATAGTGGTACTGACTC 4 56 243 MboII 146, 97 CCR2 CCTGAATAGCTGATGTGAAAG

DDF1 GATTTGTTGTTTCACTGGAGC 5 58 288 BamHI 174, 114 DDR1 TTCACTCTAGCCAGTCCTTC

EEF1 GGAGTGACTGAGCATATAAG 6 58 219 HpaII 117, 102 EER1 AACCCATCTCCAGATGTTTAG

FFF1 GCTGCAGCCAGCCTGGACC 7 60 293 PstI 167, 125 FFR1 AGTCTTCAACTTCAAGGGCATC

GGF1 ATAACTGCATTTCTCAACC 8 60 277 SfaNI 157, 120 GGR1 AAGCAAGTGGAGTGCCATC

HHF1 GGTCTGATGGGCTGAGATAC 9 60 264 FokI 145, 119 HHR1 GAGCAAGCGTCATCTTTGGAG

IIF1 CACTCCCTTGTTGTCCTCC 10 56 271 TaqI 136, 135 IIR1 TATATACACCTAGTATGTAACC

VVF1 TAAAGTGGCATACAACAGTAG 11 58 242 NcoI 131, 111 VVR1 ACCTGGCAGGGCACGCAAC

SSF1 ATTCTTTGTAGTCACCATGCC 12 58 227 HaeIII 124, 103 SSR1 AGCAAGTGTGCCGTCTCTACC

TTF1 CTACATAACATCAGTACTGCC 13 56 220 BspMI 114, 106 TTR1 TAAGACGAATGCCAGTGCC

XXF1 TACCTTTTGACTCACTGAACC 14 56 288 BclI 156, 132 XXR1 GGTACCACCTGGGTAATTC

WWF1 GAACTTTGTGTCACATGCAGC 15 56 278 FokI 160, 118 NAP3R AACATGTTTTCTCAATAAGGC

hTRF TCATGGCCGGAAATGGAACT 58 653 BstNI 229, 183,

167, 74

hTRR GGGTGACGGATGCGCACGAT

tape around the bottom of the gel Ensure that the gaskets closely abut the smaller

plate (see Note 6)

2 Take 80 mL of the SSCP gel mix Add 560 µL of 10% ammonium persulfate and

28 µL of TEMED, mix, and pour the solution slowly between the glass plates

using a 50-mL syringe (see Note 7) When full, insert an inverted sharks tooth

comb (smooth surface downward) no more than 6 mm into the gel Leave topolymerize

3 Remove the electrical tape and bulldog clips and place the gel in the phoresis tank Fill the top and bottom chambers with 1X TBE Remove the comb

electro-14 Tipping et al.

Table 6

PCR Reagent Mixes Used in Amplification of DKC1 Exons

Reagent Volume of reagent (µL) per number of reactions (N)

(see Note 8) and flush the surface of the gel with TBE buffer using a syringe and

bent needle Clean and invert the comb and insert it between the plates until theteeth just indent the surface of the gel

4 Mix 1–4 µL of the radiolabeled PCR product with 6 µL of formamide dye Heat

at 95°C for 5 min Snap chill on wet ice Flush out each well using TBE bufferand load 5 µL of each sample between the teeth Run the gel overnight at 8–12 mA

in a cool laboratory (see Note 9) As a guide, the bromophenol blue and xylene

cyanol will comigrate with approx 60 bp and 220 bp DNA fragments tively in a 6% polyacrylamide gel The single-stranded DNA fragments willmigrate considerably slower

respec-5 Disconnect the power supply, and remove the plates and place them on a flatsurface Pull one of the spacers out from between the plates Insert a metal spatula

or a fine plastic wedge horizontally into the gap between the plates at the bottomcorner where the spacer had been Lift the smaller siliconized plate off the gel.Cut a piece of 3MM Whatman paper so that it is slightly larger than the gelarea, and lay it down onto the gel Return the smaller plate over the Whatmanpaper, apply gentle pressure, and invert the plates Carefully pull up the largerplate, ensuring that the gel sticks to the Whatman paper Cover the gel with clingfilm (e.g., Saran Wrap) and trim all the edges

6 Dry the gel under vacuum at 80°C for approx 1 h Peel off the Saran Wrap andexpose the gel to X-ray film overnight at –80°C to obtain an autoradiograph

A shift in migration of the single-stranded fragment is indicative of amutation in the relevant exon Variations in the gel composition can be intro-duced to increase the chances of observing aberrant mobilities (or shifts) ofmutant DNA strands These include altering the content of the gel such as the

percent of glycerol, the percent of the acrylamide and the

acrylamide/bis-acrylamide ratio used

Molecular Diagnosis of FA and DC 15

3.6 DNA Sequencing

1 Reamplify the appropriate exon using the conditions described in Subheading 3.4.,

but scaling up the volume of the reaction to 100 µL and the cycle number to 35

2 Load the entire product onto a 1.5% agarose gel; after sufficient electrophoresis,cut out the fragment and elute the DNA using a QIAquick Gel Extraction column

3 Direct sequence analysis of the fragment is now performed by a specializedservice

qual-necessary to use more template in the PCR, paying attention to Note 3.

3 When analyzing the data with Genotyper, the peak heights in the grams should ideally be between 100 and 1000 units If significantly greater thanthis, the PCR has probably advanced beyond the exponential phase, potentiallyskewing any analysis of these results

electrophero-4 Afterassessment of which exonswere most frequently deletedfrom FANCA,

multiplex 4was developed,with exon 1 ofthe myelin protein zerogene as theexternalcontrol It is left to the reader to determine which multiplex is best suited

to his or her needs, or to develop his or her own multiplex Key in this ment is the ability to demonstrate robust amplification of all exons of interest in

develop-a vdevelop-ariety of sdevelop-amples, develop-and for develop-all develop-amplified exons to be of different sizes to develop-allowsingle-color detection during the gel run

5 If the analysis shows a single exon deletion, this result must be repeated to firm that this exon did not “drop out” (fail to amplify) during the PCR If possiblelook for deletion of other exons upstream and downstream to delimit the extent

con-of the deletion A well-designed combination con-of multiplex PCRs will allow rapidscanning of the gene for large deletions, although it should be clearly noted thatunless an exon has been examined directly, it may be deleted Such deletions are

present in FANCA in some patients, and may be missed in a scanning approach.

6 The interface between small plate and the gasket is the most likely location of aleak down from the upper chamber of the gel When the gel is set and assembled,

it is possible to dab a small amount of molten agarose into this junction to ensure

a good seal

7 The formation of bubbles as the gel is poured is a recurrent problem Concentrate

on ensuring a steady constant flow of the acrylamide solution between the plates

If they persist, hold the plates vertically and bang the bubbles to the surface If

the problem occurs repeatedly, soak the plates in 2 M NaOH for 1 h, and rinse

well with water before reassembling

8 The combs can sometimes be difficult to remove Very carefully, insert a edged razor blade between the comb and the plate and gently prise the combaway from the plate before trying to pull it out

flat-16 Tipping et al.

9 The migration of the single-stranded fragments varies considerably with the perature of the gel Our standard procedure is to air condition the lab at 18–20°C.Running the gel in a cold room (at 4°C) would represent a different set of condi-tions

tem-References

1 Auerbach, A D., Buchwald, M., and Joenje, H (1998) Fanconi anemia, in The

Genetic Basis of Human Cancer (Vogelstein, B and Kinzer, K W., eds.),

McGraw-Hill, New York, pp 317–332

2 Dokal, I (2000) Dyskeratosis congenita in all its forms Br J Haematol 110,

768–779

3 Strathdee, C A., Gavish, H., Shannon, W R., and Buchwald, M (1992) Cloning of

cDNAs for Fanconi anemia by functional complementation Nature 356, 763–767.

4 Lo Ten Foe, J R., Rooiman, M A., Bosnoyan-Collins, L., et al (1996)

Expres-sion cloning of a cDNA for the major Fanconi anemia gene, FAA Nat Genet 14,

320–323

5 The Fanconi anemia/Breast Cancer consortium (1996) Positional cloning of the

Fanconi anemia group A gene Nat Genet 14, 324–328.

6 de Winter, J P., Waisfisz, Q., Rooimans, M A., et al (1998) The Fanconi anemia

group G gene FANCG is identical with human XRCC9 Nat Genet 20, 281–283.

7 de Winter, J P., Rooimans, M A., van der Weel, L et al (2000) The Fanconianemia complementation gene FANCF encodes a novel protein with homology to

ROM Nat Genet 24, 15–16.

8 de Winter, J P., Leveille, F., van Berkel, C G M., et al (2000) Isolation of a

cDNA representing the Fanconi anemia complementation group E gene Am J.

Hum Genet 67, 1306–1308.

9 Timmers, C., Taniguchi, T., Hejna, J., et al (2001) Positional cloning of a novel

Fanconi anemia gene, FANCD2 Mol Cell 7, 241–248.

10 Morgan, N V., Tipping, A J., Joenje, H., and Mathew, C G (1999) High

fre-quency of large intragenic deletions in the Fanconi anemia group A gene Am J.

Hum Genet 65, 1330–1341.

11 Verlander, P C., Lin, J D., Udono, M U., et al (1994) Mutation analysis of the

Fanconi anemia gene FACC Am J Hum Genet 54, 595–601.

12 Gibson, R A., Morgan, N V., Goldstein, L H., et al (1996) Novel mutations and

polymorphisms in the Fanconi anemia group C gene Hum Mutat 8, 140–148.

13 Yau, S C., Bobrow, M., Mathew, C G., and Abbs, S J (1996) Accurate sis of carriers of deletions and duplications in Duchenne/Becker muscular dystro-

diagno-phy by fluorescent dosage analysis J Med Genet 33, 550–558.

14 Heiss, N S., Knight, S W., Vulliamy, T J., et al (1998) X-linked dyskeratosiscongenita is caused by mutations in a highly conserved gene with putative nucle-

olar functions Nat Genet 19, 32–38.

15 Vulliamy, T., Marrone, A., Goldman, F., et al (2001) The RNA component of

telomerase is mutated in autosomal dominant dyskeratosis congenita Nature 413,

432–435

Molecular Diagnosis of FA and DC 17

16 Knight, S W., Heiss, N S., Vulliamy, T J., et al (1999) X-linked dyskeratosis

congenita is predominantly caused by missense mutations in the DKC1 gene.

Am J Hum Genet 65, 50–58.

17 Mullenbach, R., Lagoda P J., and Welter, C (1989) An efficient salt–chloroform

extraction of DNA from blood and tissues Trends Genet 5, 391.

Molecular Diagnosis of Diamond–Blackfan Anemia

Sarah Ball and Karen Orfali

Introduction

1.1 Clinical Features of Diamond–Blackfan Anemia (DBA)

Diamond–Blackfan anemia (DBA) is a rare congenital pure red cell aplasia,

with an incidence of 4–7 per million live births (1–5) Typically, affected

chil-dren present in the second or third month of life with profound anemia, often in

association with craniofacial (6) or thumb anomalies (2,7), and small stature

(2) In 15–20% there is a positive family history, characterized by an

autoso-mal dominant pattern of inheritance (2–4) In the majority of cases, the anemia

is responsive to steroids, but eventually up to 40% of affected individuals aredependent on a life-long transfusion program, unless they undergo successful

stem cell transplantation (2,3,8) Spontaneous remission may occur, although

this is unpredictable In the longer term, there is an increased risk of

myelodys-plasia and myeloid leukemia (9), and probably also of other malignancies (10).

1.2 Hematological Parameters of DBA

The blood cell count at presentation is characterized by anemia andreticulocytopenia, with isolated marrow erythroid hypoplasia The mean cellvolume is usually within the normal range for infants, but is generally raised in

children presenting at an older age (9) During steroid-induced or spontaneous remission, there is usually a persistent mild macrocytic anemia (2,11,12), often

with raised fetal hemoglobin (HbF), and persistent strong expression of the

blood group antigen i (12,13), which is usually only weakly expressed beyond

infancy

20 Ball and Orfali

1.3 Differential Diagnosis of DBA: Potential Applications

for a Molecular Diagnostic Approach

The differential diagnosis of red cell aplasia presenting in infancy is rily between an early presentation of transient erythroblastopenia of childhood

prima-(TEC) (14–16) and chronic parvovirus infection (17–19) The diagnosis of an

acquired immune-mediated pure red cell aplasia should also be considered inolder children

In individuals with “classical” DBA as outlined, the diagnosis may beunequivocal However, for children with less typical features, especially thosepresenting at an older age, the diagnosis may be less clear cut As chronicparvoviremia is associated with a failure to mount an appropriate immune

response (17–19), there may not be serological evidence of parvovirus, and

parvovirus infection should be excluded by direct detection of parvovirus

DNA (20).

Misdiagnosing other causes of childhood red cell aplasia as DBA may notonly result in inappropriate treatment, but can also induce unnecessary familialanxiety, as the diagnosis of DBA carries genetic and other longer term implica-

tions (9,10) A molecular diagnostic approach is therefore of potential value in

the differential diagnosis of red cell aplasia in childhood It also has otherimportant applications; in genetic counseling, in the exclusion of subclinical

DBA in sibling donors when planning stem cells transplantation (BMT) (21),

and in the detailed characterization of probands and other family members for

genetic linkage studies (13,22).

1.4 Erythrocyte Adenosine Deaminase Activity

Currently the most useful investigation in the molecular diagnosis of DBA

is the measurement of erythrocyte adenosine deaminase (eADA) activity,

which is raised in the great majority of patients with DBA (11–13,23–27) Its

value as a diagnostic tool is limited in patients who are transfusion dependent.Although measurement as late as possible posttransfusion may reveal eADA

activity above the normal range (unpublished observations), a normal level is

uninterpretable Cord blood eADA activity falls within the normal reference

range (23,25,28), making eADA a potentially useful tool to screen for DBA in

the newborn siblings of affected children

1.4.1 Interpretation of Results:

Differential Diagnosis of Raised eADA Activity

Raised eADA activity is not specific for DBA, although it is still useful in

discriminating between DBA and TEC (25) It has also been described in paroxysmal nocturnal hemoglobinuria (PNH) (29), myelodysplastic syndromes

Diamond–Blackfan Anemia 21

(MDS) (29,11,23), and Down’s syndrome (30); interestingly, like DBA, all of

these disorders are associated with macrocytosis

1.4.2 eADA Measurement in Family Studies

Several studies have shown that eADA activity may also be raised in some

first degree relatives of patients with DBA (13,23,26,27,31) In families with

RPS19 mutations (see below), raised eADA activity usually, but not always,

cosegregates with the family RPS19 mutation Isolated raised eADA in degree relatives has also been demonstrated in families without RPS19 muta-

first-tions Although 15–20% have an unequivocal family history of DBA (2–4),

detailed family studies, including measurement of eADA activity, have

revealed a wider range of phenotypic expression of DBA (13,27) The cosegregation of RPS19 mutations with increased eADA activity (13) has led

to the realization that family members with high eADA activity, with or out mild anemia or macrocytosis, should be considered as having a subclinical

with-or silent fwith-orm of DBA (13,27) It is often possible in such families to elicit a

history of unexplained self-remitting anemia in early childhood or during

preg-nancy (13,23,27) However, the broadening of the accepted DBA phenotype to

include isolated high eADA activity does challenge the accepted classical

diagnostic criteria for DBA (32) The other conclusion to be drawn from these

family studies is that fewer cases of DBA are truly sporadic than originallybelieved; the proportion of these is possibly as low as 50%

1.4.3 Principle of Erythrocyte Adenosine Deaminase Assay

ADA catalyzes the hydrolytic deamination of adenosine to produce inosineand ammonia The method described here is a continuous spectrophotometricassay, based on the difference in the extinction coefficients of adenosine and

inosine at 265 nm (33,34) It is convenient, inexpensive, and entails no

expo-sure to radioactivity However, it must be stressed that it is not designed for the

diagnosis of ADA deficiency Radioisotopic (28) or radioimmunoassay (35)

methods are also available, while coupled enzyme assays can be used to

mea-sure the production of ammonia (36), or the conversion of inosine to xanthine and uric acid (37).

1.5 Genetics of DBA: Direct Detection of Mutation

1.5.1 Gene Encoding Small Ribosomal Protein 19 (RPS19)

Most patients with DBA have normal cytogenetics, but the serendipitousfinding of a child with sporadic DBA and a balanced chromosomal transloca-

tion t(X;19) (38) led to the identification of RPS19 as the first “DBA gene”

(39) Twenty-five percent of individuals with DBA have been found to have

22 Ball and Orfali

mutations affecting a single allele of RPS19 (40), consistent with the observed

autosomal dominant pattern of inheritance Direct detection of a mutation

affecting RPS19 thus leads to a definitive diagnosis of DBA, although the wide

variation in phenotype observed between individuals with identical mutations,

even within the same family (41,42), should lead to caution in genetic

counsel-ing There may also be difficulty in differentiating between polymorphismsand functionally significant mutations affecting noncoding sequences, espe-cially if the control population and the family with the putative mutation arefrom divergent ethnic backgrounds The method described here is that of directsequencing of PCR products to include all coding regions, intron–exon bound-

aries, and upstream noncoding regions (39–41,43) This approach may of

course miss total allele loss or large deletions, which might be detected bySouthern blot or loss of heterozygosity of linked polymorphisms In the origi-

nal 19q13 linkage study (44), one sporadic case (of 13 studied) had evidence of

loss of heterozygosity of 19q13 markers, the result of a large deletion that

included one RPS19 allele Large deletions encompassing RPS19 may be

associated with more severe skeletal anomalies and mental retardation,

sug-gesting a contiguous gene syndrome (45) However, such total allele deletions

do not appear to be common, making it unlikely that many RPS19 deletions

will be missed by using polymerase chain reaction (PCR) and direct ing In addition, the demonstration of heterozygosity for intragenic polymor-phisms rules out total loss of one allele, while the clear lack of cosegregation

sequenc-between DBA and 19q in the majority of families (22,46), despite the high logarithm of odds (LOD) score for 19q13 in the original linkage study (44), is consistent with RPS19 mutations accounting for only 25% of cases (40).

1.5.2 Other DBA Genes

Significant linkage has been established to chromosome 8p in 47% of cases

of familial DBA, but the gene responsible has not yet been identified (22) In

addition, the observed failure of linkage to both 19q and 8p in up to 20% offamilies with a clear pattern of inheritance demonstrates the existence of at

least a third DBA gene (22) Thus there is currently no way to diagnose DBA at

the genetic level in the 75% of patients without an RPS19 mutation.

2 Materials

2.1 Erythrocyte Adenosine Deaminase Assay

2.1.1 Preparation of Lysate (see Notes 1 and 2)

1 Blood (in EDTA)

2 Normal saline: 0.9% NaCl in water

3 Lysate stabilizer (47): 2.7 mM EDTA, pH 7.0, 0.7 mM β-mercaptoethanol Store

at 4°C

Diamond–Blackfan Anemia 23

2.1.2 ADA Asay

1 Distilled water

2 ADA assay buffer: 1 M Tris-HCl, 5 mM EDTA, pH 8.0, store at 4°C

3 4 mM Adenosine (Sigma): Make up in lysate stabilizer and store at –20°C insmall aliquots

4 Drabkin’s solution (Sigma)

5 Cuvets suitable for wavelength (e.g., silica from Merck)

6 Ultraviolet (UV)/visible spectrophotometer with heated carousel (30°C) (e.g.,

Ultrospec range from Amersham–Pharmacia) (see Note 3).

7 Enzyme kinetics application software for spectrophotometer (see Note 4).

2.2 RPS19 PCR for Sequencing

1 Genomic DNA to be sequenced, 200 ng/PCR reaction

2 Nuclease-free water

3 Primers at a working concentration of 10 pmol/µL (see Table 1)

4 Hot start DNA polymerase (AmpliTaq Gold; Applied Biosystems)

5 10X PCR buffer containing 15 mM MgCl2 (supplied with AmpliTaq Gold;Applied Biosystems)

6 dNTP mix: 2.5 mM of each deoxynucleoside triphosphate.

7 5X Q-Solution (Qiagen)

8 Mineral oil (Sigma) (see Note 5).

9 Thermocycler

10 Equipment for agarose gel electrophoresis

11 10X TAE electrophoresis buffer: 0.4 M Tris-acetate, pH 8.0, 10 mM EDTA.

24 Ball and Orfali

3 Method

3.1 Preparation of Lysate (47)

1 Wash 100 µL of whole blood twice with normal saline (see Notes 6 and 7):

a Suspend cells in saline in a 15-mL polypropylene tube

b Centrifuge at 1000g for 5 min.

c Remove as much supernatant as possible

d Repeat

2 Add 500 µL of lysate stabilizer to cell pellet and vortex mix

3 Rapid-freeze to ensure complete lysis, using one of the following methods:

a In a –70°C freezer for 1–2 h (see Note 8).

b In methanol prechilled to –20°C

c In dry-ice–acetone bath

4 Thaw in a 25°C water bath

5 Transfer to ice as soon as thawed (see Note 9).

3.2 ADA Assay

1 Take an aliquot of 4 mM adenosine from the freezer and thaw on ice.

2 Switch on the spectophotometer and water bath

3 Pipet into silica cuvets and mix by inversion or stirring

a 860 µL of distilled water

b 100 µL of ADA assay buffer

c 20 µL of hawed lysate

4 Place cuvets in a heated carousel at 30°C

5 Incubate for 10 min before starting the assay

6 Blank against first test cuvet before adding adenosine (see Note 10).

7 Start the assay by adding 20 µL of 4 mM adenosine and mix by inversion.

8 Follow reaction for 19 min, recording A265 every 30 s

6 Record the slope (∆A265) and linearity for the last 5 min (see Note 11).

3.3 Measurement of Hb Concentration of Lysate

1 Add 20 µL of lysate to 980 µL of Drabkin’s solution

2 Allow to stand for 5 min before reading absorbance

3 Read absorbance at 540 nm against Drabkin’s blank

4 Calculate the hemoglobin concentration of the lysate using the formula:

Hb (g/dL) = A540× 7.47

3.4 Calculation of ADA Activity (see Notes 12–15)

eADA (U/gHb) = ∆A265× 5000

8.1× Hb (g/dL)

Diamond–Blackfan Anemia 25

3.5 RPS19 PCR Reactions

3.5.1 Set 1

In a 500-µL PCR tube, combine:

a Nuclease-free water to a total volume of 50 µL

b 200 ng of template DNA (see Note 16).

h 1.25 U of hot start Taq polymerase.

i Overlay with a few drops of mineral oil (see Note 18).

3.5.2 Sets 2–4

As for Set 1, but with the omission of Q solution, with relevant primers for

the appropriate set

3 Extend at 72°C for 10 min

4 Purify immediately or store at –20°C until needed

3.6 Gel Purification of PCR Products

1 Separate PCR products on 1% TAE-agarose gel (see Note 21).

2 Visualize ethidium bromide stained gel on UV transilluminator

3 Excise bands (see Notes 22–24).

4 Purify bands with Qiagen QIAquick Gel Extraction Kit according to the facturer’s instructions

manu-5 Elute purified DNA in the minimum recommended volume to avoid overdilution

for sequencing (see Notes 25–27).

4 Notes

1 This is a whole blood method; there is no need for a white blood cell depletion step

2 Stable for up to 72 h at room temperature (11), and up to 1 wk at 4 °C (25).

3 We use Ultrospec III, which is adequate for the purpose but now newer modelsare available

4 Not essential, as manual recording of a fall in absorbance is an alternative (albeitlaborious) option

26 Ball and Orfali

5 Not needed if using a heated lid

6 If very anemic increase the volume of whole blood to give a minimum estimatedpacked red blood cell volume of 30 µL

7 Include a normal control in each batch

8 Maximum overnight

9 Activity is stable on ice for several hours but not overnight

10 As reaction causes a fall in OD, blanking after adding adenosine will cause the

OD to drop into the negative range

11 Often nonlinear at start

12 Based on 8.1 = difference in millimolar extinction coefficient of adenosine andinosine One unit is defined as the activity that catalyzes deamination of 1 mmoladenosine per minute under the assay conditions

13 ∆A265 is usually in the range –0.002 to 0.004/min for normals

14 Established normal range in our laboratory (n = 33): mean ± SD = 0.605 ± 0.198

(27) Upper limit as defined by normal mean + 2 SD = 1.00 U/g Hb In a study of

28 transfusion-independent patients with DBA, the mean eADA was 2.631 ±

1.873 (p < 0.0001) (27).

15 Collaborative European studies, to allow for differences in methods between ters, generally express patient eADA in terms of number of SD from mean forthat method

cen-16 Include negative control tube with no DNA for each set of primers in each batch

of reactions

17 Needed because of high GC content and secondary structure

18 Unless using a cycler with a heated lid

19 This is essential if using a hot start polymerase, as it both denatures misprimingthat may have occurred during setup and activates the enzyme

20 These conditions are good in our laboratory for all the primer sets, allowing all to

be amplified using the same conditions, but modification may be necessary tooptimize the PCR product for different thermocyclers

21 Run 3 µL if simply confirming success of reaction For purification, run the entirecontents of the PCR reaction

22 See Table 1 for band sizes.

23 Primer set 2 often generates a second minor band, migrating more slowly

24 Cut as close to the bands as possible to minimize the presence of excess agarosefor the gel extraction procedure

25 Methods for sequencing are not included here For a laboratory that does notroutinely undertake sequencing, it is usually more cost effective to use a com-mercial sequencing service, using their recommended primer and template con-centrations

26 The PCR primers are also suitable for sequencing Further primer sequences, forexample, for confirmation of a mutation, can be derived from the genomic

sequence of RPS19 (GenBank accession numbers AF092906 and AF092907) (39).

27 Primer set 2 encompasses an insertion/deletion polymorphism in intron 2, whichgenerates a multiple sequencing reaction downstream of the polymorphism in

Diamond–Blackfan Anemia 27

heterozygotes The heterozygosity rate is approx 50% (unpublished

observa-tions) There is also a linked single nucleotide polymorphism in intron 2, and

another in intron 4

References

1 Bresters, D., Bruin, M C A., and Van Dijken, P J (1991) Congenitale

hypoplastiche anemia in Nederland (1963–1989) Tijdschrift Kindergen 59, 203–210.

2 Ball, S E., McGuckin, C P., Jenkins, G., and Gordon-Smith, E C (1996)Diamond–Blackfan anemia in the UK: analysis of 80 cases from a 20-yr birth

cohort Br J Haematol 94, 645–653.

3 Willig, T N., Niemeyer, C M., Leblanc, T., et al (1999) Identification of newprognosis factors from the clinical and epidemiologic analysis of a registry of 229

Diamond–Blackfan anemia patients Pediatr Res 46, 553–561.

4 Ramenghi, U., Garelli, E., Valtolina, S., et al (1999) Diamond–Blackfan anemia

in the Italian population Br J Haematol 104, 841–848.

5 Vlachos, A., Klein, G W., and Lipton, J M (2001) The Diamond Blackfan mia Registry: tool for investigating the epidemiology and biology of Diamond–

Ane-Blackfan anemia J Pediatr Hematol Oncol 23, 377–382.

6 Cathie, I A B (1950) Erythrogenesis imperfecta Arch Dis Child 25, 313–324.

7 Aase, J M and Smith, D W (1969) Congenital anemia and triphalyngeal thumbs:

a new syndrome J Pediatr 74, 471–474.

8 Vlachos, A., Federman, N., Reyes-Haley, C., Abramson, J., and Lipton, J M.(2001) Hematopoietic stem cell transplantation for Diamond–Blackfan anemia: a

report from the Diamond–Blackfan Anemia Registry Bone Marrow Transplant

27, 381–386.

9 Janov, A J., Leong, T., Nathan, D G., and Guinan, E C (1996) Diamond

Blackfan anemia Natural history and sequelae of treatment Medicine 75, 77–78.

10 Lipton, J M., Federman, N., Khabbaze, Y., et al (2001) Osteogenic sarcomaassociated with Diamond–Blackfan anemia: a report from the Diamond–Blackfan

Anemia Registry J Pediatr Hematol Oncol 23, 39–44.

11 Glader, B E and Backer, K (1988) Elevated red cell adenosine deaminase ity: a marker of disordered erythropoiesis in Diamond–Blackfan anemia and other

activ-haematologic diseases Br J Haematol 68, 165–168.

12 Whitehouse, D B., Hopkinson, D A., and Evans, D I K (1984) Adenosine

deaminase activity in Diamond–Blackfan syndrome Lancet 2, 1398–1399.

13 Willig, T N., Perignon, J L., Gustavsson, P., et al (1998) High adenosinedeaminase level among healthy probands of Diamond Blackfan Anemia (DBA)

cosegregates with the DBA gene region on chromosome 19q13 Blood 92,

16 Kynaston, J A., West, M C., and Reid, M M (1993) A regional experience of

red cell aplasia Eur J Pediatr 152, 306–308.

28 Ball and Orfali

17 Kurtzmann, G J., Ozawa, K., Cohen, B., Hanson, G., Oseas, R., and Young, N S.(1987) Chronic bone marrow failure due to persistent parvovirus B19 infection

N Engl J Med 317, 287–294.

18 Frickhofen, N and Young, N S (1989) Persistent parvovirus B19 infection in

humans Microb Pathog 7, 319–327.

19 Kurtzmann, G J., Cohen, B J., Field, A M., Oseas, R., Blaese, R M., and Young,

N S (1989) Immune response to B19 parvovirus and an antibody defect in

persis-tent viral infection J Clin Invest 84, 1114–1123.

20 Salimans, M M., Holsappel, S., van de Rijke, F M., Jiwa, N M., Raap, A K., andWeiland, H T (1989) Rapid detection of human parvovirus B19 by dot-hybrid-

ization and the polymerase chain reaction J Virol Methods 23, 19–28.

21 Orfali, K A., Wynn, R F., Stevens, R F., Chopra, R., and Ball, S E (1999)Failure of red cell production following allogeneic BMT for Diamond Blackfananemia (DBA) illustrates the functional significance of high erythrocyte adenos-

ine deaminase (eADA) activity in the donor (abstract) Blood 94 (Suppl 1), 414a.

22 Gazda, H., Lipton, J M., Willig, T N., et al (2001) Evidence for linkage of ial Diamond-Blackfan anemia to chromosome 8p23.3-p22 and for non-19q non-

famil-8p disease Blood 97, 2145–2150.

23 Glader, B E., Backer, K., and Diamond, L K (1983) Elevated erythrocyte

adenosine deaminase activity in congenital hypoplastic anemia N Engl J Med.

309, 1486–1490.

24 Whitehouse, D B., Hopkinson, D A., Pilz, A J., and Arrecondo, F X (1986)Adenosine deaminase activity in a series of 19 patients with the Diamond-

Blackfan syndrome Adv Exp Med Biol 195, 85–92.

25 Glader, B E and Backer, K (1986) Comparative activity of erythrocyte adeonsine

deaminase and orotidine decarboxylase in Diamond-Blackfan syndrome Am J.

apparently sporadic cases of DBA Br J Haemat 105 (Suppl 1), 72.

28 Perignon, J L., Durandy, A., Peter, M O., Freycon, F., Dumez, Y., and Griscelli,

C (1987) Early prenatal diagnosis of inherited severe immunodeficiencies linked

to enzyme deficiencies J Pediatr 111, 595–598.

29 di Marco, P., Tinnirello, D., Tambone-Reyes, M., Tedesco, L., Luna, S., andCitarella, P (1992) Biologic relevance of elevated red cell adenosine deaminaseactivity in myelodysplastic syndromes and paroxysmal nocturnal hemoglobinuria

Tumori 78, 370–373.

30 Ibarra, B., Rivas, F., Medina, C., et al (1990) Hematological and biochemical

studies in children with Down syndrome Ann Genet 33, 84–87.

31 Filanovskaya, L I., Nikitin, D O., Togo, A V., Blinov, M N., and Gavrilova, L

V (1993) The activity of purine nucleotide degradation enzymes and lymphoid

Diamond–Blackfan Anemia 29

cell subpopulation in children with Diamond–Blackfan syndrome Gematol.

Transfuziol 38, 19–22.

32 Willig, T N., Ball, S E., and Tchernia, G (1998) Current concepts and issues in

Diamond-Blackfan anemia Curr Opin Hematol 5, 109–115.

33 Kalckar, H M (1947) Differential spectrophotometry of purine compounds bymeans of specific enzymes III Studies of the enzymes of purine metabolism

J Biol Chem 167, 461–475.

34 Agarwal, R P., Sagar, S M., and Parks, R E J (1975) Adenosine deaminasefrom human erythrocytes: purification and effects of adenosine analogues

Biochem Pharmacol 24, 693–701.

35 Daddona, P E., Frohman, M A., and Kelley, W N (1981) Radioimmunoassay of

human adenosine deaminase Methods Enzymol 74, 351–358.

36 Agarwal, R.P., Crabtree, G W., Parks, R E Jr., et al (1976) Purine nucleosidemetabolism in the erythrocytes of patients with adenosine deaminase deficiency

and severe combined immunodeficiency J Clin Invest 57, 1025–1035.

37 Hopkinson, D A., Cook, P J L., and Harris, H (1969) Further data on the

adenosine deaminase (ADA) polymorphism and a report of a new phenotype Ann.

Hum Genet 32, 361–367.

38 Gustavsson, P., Skeppner, G., Johansson, B., et al (1997) Diamond–Blackfan

anemia in a girl with a de novo balanced reciprocal X;19 translocation J Med.

Genet 34, 779–782.

39 Draptchinskaia, N., Gustavsson, P., Andersson, B., et al (1999) The gene

encod-ing ribosomal protein S19 is mutated in Diamond–Blackfan anemia Nat Genet.

21, 169–175.

40 Willig, T N., Draptchinskaia, N., Dianzani, I., et al (1999) Mutations in mal protein S19 gene and Diamond Blackfan anemia: wide variations in pheno-

riboso-typic expression Blood 94, 4294–4306.

41 Ramenghi, U., Campagnoli, M F., Gerelli, E., et al (2000) Diamond–Blackfananemia: report of seven further mutations in the RPS19 gene and evidence of

mutation heterogeneity in the Italian population Blood Cells Mol Dis 26,

417–422

42 Matsson, H., Klar, J., Draptchinskaya, N., et al (1999) Truncating ribosomalprotein S19 mutations and variable clinical expression in Diamond–Blackfan ane-

mia Hum Genet 105, 496–500.

43 Cmejla, R., Blafkova, J., Stopka, T., et al (2000) Ribosomal protein S19 tions in patients with Diamond–Blackfan anemia and identification of ribosomal

muta-protein S19 pseudogenes Blood Cells Mol Dis 26, 124–132.

44 Gustavsson, P., Willig, T N., van Haeringen, A., et al (1997) Diamond–Blackfananemia: genetic homogeneity for a gene on chromosome 19q13 restricted to

30 Ball and Orfali

46 Gustavsson, P., Garelli, E., Draptchinskaia, I., et al (1998) Identification ofmicrodeletions spanning the Diamond–Blackfan anemia (DBA) locus on 19q13

and evidence for genetic heterogeneity Am J Hum Genet 63, 1388–1395.

47 Beutler, E., Blume, K G., Kaplan, J C., Lohr, G W., Ramot, B., and Valentine, W

N (1977) International Committee for Standardization in Haematology:

recommended methods for red-cell enzyme analysis Br J Haematol 35, 331–340.

Antenatal Diagnosis of Hemoglobinopathies 33

analysis (1,2) Each method has its advantages and disadvantages, and the

particular one chosen by a laboratory to diagnose point mutations depends notonly on the technical expertise available in the diagnostic laboratory but also

on the type and variety of the mutations likely to be encountered in the viduals being screened

indi-Prenatal diagnosis of β-thalassemia was first accomplished in 1974, andsince then many countries have developed an extremely successful programfor controlling the disorder based on population screening and fetal diagnosis.Initially this was performed by the measurement of globin chain synthesis infetal blood, obtained by fetal blood sampling at 18–20 wk of gestation How-ever DNA analysis techniques soon began to replace the globin chain synthesisapproach, first by the indirect technique of restriction fragment length poly-morphism (RFLP) analysis, then by direct detection of mutations by restrictionenzyme digestion and later by oligonucleotide hybridization to DNA fragments

on a Southern blot All of these Southern blot based technique were complexand expensive and prenatal diagnosis remained inaccessible for developing

34 Oldcountries until the discovery of PCR, which led to the development of simpler,quicker, and less expensive nonradioactive methods of mutation detection.Fetal DNA was obtained from cultured amniotic fluid cells until 1982, whenchorionic villus sampling (CVS) in the first trimester of pregnancy was devel-

oped (3) Currently, prenatal diagnosis by CVS DNA analysis is the method of

choice because it is carried out at the 10th to 12th wk of gestation, the risk offetal mortality associated with the method is acceptably low at 1%, and suffi-cient DNA is obtained for analysis without cell culture

1.1 Identification of At-Risk Couples

Couples at risk for severe hemoglobin disorders are first identified by tological screening tests as directed by published guidelines and flow charts

hema-(4) The basic tests are the measurement of the mean corpuscular volume

(MCV) and mean corpuscular hemoglobin (MCH) values, the levels of HbA2and HbF, and the detection of abnormal hemoglobins by electrophoresis meth-ods or high-performance liquid chromatography (HPLC) An individual with areduced MCV and MCH with a normal HbA2 level has α-thalassemia; an indi-vidual with a raised HbA2 level has β-thalassemia, and one with a raised HbFlevel of 5–15% has δβ-thalassemia An individual with normal red cell indicesand a HbF level of 15–30% has hereditary persistence of fetal hemoglobin(HPFH) This approach has many pitfalls, however, that may lead to a wrongcarrier identification These include: the presence of iron deficiency, whichalso reduces the MCV and MCH; mild β-thalassemia mutations, which areassociated with borderline raised HbA2 levels; and the coinheritance of aδ-thalassemia mutation, which reduces the HbA2 level in an individual with

β-thalassemia trait to a normal value (5) Certain carrier combinations can give

rise to a hidden risk (i.e., a risk not easily discernible by simple hematologicalanalysis) of having a fetus affected with homozygous α0-thalassemia Thisoccurs because the carrier state for various β-thalassemia disorders can maskcoexisting α0-thalassemia trait in regions where both disorders are found (e.g.,

in Southeast Asia) Thus for couples in which one partner is diagnosed byhematological screening as a carrier of β-thalassemia and the other a possiblecarrier of α0-thalassemia, the individual with β-thalassemia should also bescreened for α0-thalassemia mutations by DNA analysis

1.2 Diagnostic Approaches to Mutation Screening

The main diagnostic approaches for the PCR diagnosis of the

hemoglobino-pathies are listed in Table 1 The ones well used in my laboratory are

gap-PCR, ARMS-gap-PCR, and RE analysis of amplified product Detailed protocolsfor each of these techniques are presented in this chapter The alternative well

Antenatal Diagnosis of Hemoglobinopathies 35

Table 1

DNA Diagnosis of the Hemoglobinopathies

Disorder and mutation Type Diagnostic method

α0-Thalassemia Gap-PCR, Southern blotting

α+-Thalassemia

Deletion Gap-PCR, Southern blotting

Nondeletion ASO, RE, DGGE

β-Thalassemia

Deletion Gap-PCR

Nondeletion ASO, ARMS, DGGE

δβ-Thalassemia Gap-PCR, Southern blotting

HPFH

Deletion Gap-PCR, Southern blotting

Nondeletion ASO, ARMS, RE, DGGE

Hb Lepore Gap-PCR

HbD Punjab ASO, ARMS, RE

HbO Arab ASO, ARMS, RE

Hb variants RT-PCR and DNA sequencing

ASO, allele-specific oligonucleotide hybridization; ARMS,

amplifi-cation refractory mutation system; DGGE, denaturing gradient gel

electroporesis; Gap-PCR, nutagenically separated polymerase chain

reac-tion; Hb, hemoglobin; HPFH, hereditary persistence of fetal hemoglobin;

RE, restriction endonuclease analysis.

used method for the diagnosis of hemoglobin mutations, that of allele-specificoligonucleotide (ASO) hybridization by dot blotting or reverse dot blotting, is

not covered in this chapter but detailed protocols may be found elsewhere (5).

1.2.1 α-Thalassemia

Gap-PCR provides a quick diagnostic test for α+-thalassemia and α0semia deletion mutations but requires careful application for prenatal diagno-sis Most of the common α-thalassemia alleles that result from gene deletionscan be diagnosed by gap-PCR Primer sequences have now been published forthe diagnosis of five α0-thalassemia deletions and two α+-thalassemia dele-

-thalas-36 Old

Table 2

Halassemia Deletion Mutations That Have Been Diagnosed by Gap-PCR

Disorder Deletion mutation Reference

Amplification of sequences in the α-globin gene cluster is technically moredifficult than that of the β-globin gene cluster, requiring more stringent condi-tions for success owing to the higher GC content of the breakpoint sequences

Antenatal Diagnosis of Hemoglobinopathies 37and the considerable sequence homology within the α-globin gene cluster.Experience in many laboratories has shown some primer pairs to be unreliable,resulting occasionally in unpredictable reaction failure and the problem of

allele dropout The more recently published primers (8,9), however, seem to be

much more robust at amplifying than the earlier published sequences, possiblyowing to the addition of betaine to the reaction mixture They are also designedfor a multiplex screening test, although in my laboratory they are still used inpairs to test for individual mutations

The other α0- and α+-thalassemia mutations cannot be diagnosed by PCRbecause their breakpoint sequences have not been determined These deletionmutations are diagnosed by the Southern blot technique using ζ-gene andα-gene probes This approach is still very useful, as it permits the diagnosis

of α-thalassemia deletions and α-gene rearrangements (the triple and quadruple

α-gene alleles) in a single test (10) α+-Thalassemia is also caused by pointmutations in one of the two α-globin genes These nondeletion alleles can bedetected by PCR using a technique of selective amplification of each α-globin

gene followed by a general method of mutation analysis such as DGGE (11) or DNA sequence analysis (12) Several of the nondeletion mutations alter a

restriction enzyme site and may be diagnosed by selective amplification andrestriction endonuclease analysis in a manner similar to that reported for themutation that gives rise to the unstable α-globin chain variant Hb Constant

Spring (13).

1.2.2 β-Thalassemia

The β-thalassemia disorders area very heterogeneous group of defects with

more than 170 different mutations characterized to date (14) The majority of

the defects are single nucleotide substitutions, insertions, or deletions Only 13large gene deletions have been identified, and eight of these can be diagnosed

by gap-PCR, as listed in Table 2 Different methods are required for the

detec-tion of other mutadetec-tions although the basic principles are the same That is tosay that mutation is region specific and each at-risk population has a few com-mon mutations together with a larger variable number of rare ones Thus forany given ethnic region a PCR method designed to detect the common specificmutations simultaneously is employed initially Such an approach will identifythe mutation in more than 80% of cases for most ethnic groups Further screen-ing of the known rare mutations will identify the defect in another 10–15% ofcases if necessary Mutations remaining unidentified at this stage are charac-terized by DNA sequencing

The first PCR-based method to gain widespread use was the hybridization

of ASO to amplified DNA bound to nylon membrane by dotblotting (15).

38 OldAlthough still in use, the method is limited by the need for separate hybridiza-tion steps to test for multiple mutations This was overcome by the develop-ment of the reverse dotblotting technique, in which amplified DNA ishybridized to a panel of mutation-specific probes fixed to a nylon strip Thistechnique is compatible with the optimum strategy for screening β-thalassemiamutations, using a panel of the commonly found mutations for the first screen-

ing and a panel of rare ones for the second screen (16).

My laboratory uses the ARMS (10) This fulfils the main requirements of a

PCR technology—speed, cost, convenience, and the ability to test for multiplemutations simultaneously No labeling of primers or amplified DNA isrequired The simplest approach is to screen for mutations with simultaneousPCR assays although the multiplexing of ARMS primers in a single PCR assay

is possible (17).

DGGE (18) is the most widely used indirect method to characterize semia mutations This detects at least 90% of β-thalassemia mutations by ashifted band pattern to normal and provides an alternative approach to ASOprobes or ARMS in countries where a very large spectrum of β-thalassemia

β-thalas-mutations occur (19).

1.2.3 δβ-Thalassemia and HPFH

δβ-Thalassemia and the HPFH disorders result from large gene deletionsaffecting both the β- and δ-globin genes Restriction enzyme mapping hasenabled the characterization of more than 50 different deletions starting at dif-ferent points between the Gγ-gene and the δ-gene and extending up to 100 kbdownstream of the β-globin gene In two cases, the Macedonian/Turkish (δβ)0-thalassemia gene and the Indian (Αγδβ)0-thalassemia gene, the mutation is acomplex rearrangement consisting of an inverted DNA sequence flanked bytwo deletions The breakpoint sequences have been characterized in small num-

ber of these deletions and these can be diagnosed by gap-PCR (20) Gap-PCR

can also be used for the diagnosis of the Hb Lepore, created by a deletion of theDNA sequence between the δ- and β-globin genes Hb Lepore is the product ofthe δβ fusion gene, and is associated with a severe β-thalassemia phenotype.All the deletion mutations currently diagnosable by gap-PCR are listed in

Table 2; the others can be diagnosed only by the identification of

characteris-tic breakpoint fragments with Southern blot analysis

1.2.4 Hb Variants

More than 700 hemoglobin variants have been described to date, most ofwhich were identified by protein analysis and have never been characterized atthe DNA level Positive identification at the DNA level is achieved by selec-

Antenatal Diagnosis of Hemoglobinopathies 39tive globin gene amplification and DNA sequence analysis However, the clini-cally important variants, HbS, HbC, HbE, HbD Punjab and HbO Arab, can bediagnosed by simpler DNA analysis techniques All these variants can be diag-nosed by ASO hybridization, the ARMS technique, or, except HbC, by restric-

tion endonuclease digestion of the PCR product (10) The sickle-cell gene

mutation abolishes a DdeI recognition site at codon 6 and diagnosis by DdeI

digestion of amplified product remains the simplest method of DNA analysis

or sickle-cell disease Similarly, the mutations giving rise to HbD Punjab and

HbO Arab abolish an EcoRI site and at codon 121 However, the HbC tion at codon 6 does not abolish the DdeI site and is diagnosed by other meth-

muta-ods HbE interacts with β-thalassemia trait to produce a clinical disorder ofvarying severity ranging from thalassemia intermedia to transfusion-depen-dent thalassemia major The HbE mutation can be diagnosed by ASO hybrid-

ization, ARMS, or RE analysis as the mutation abolishes a MnlI site in the

β-globin gene sequence

1.3 Indications for Antenatal Diagnosis

Antenatal diagnosis is indicated for homozygous α0-thalassemia, gous β-thalassemia, HbSS disease, and various compound heterozygous statesfor globin gene mutations that interact to result in a severe clinical disorder as

homozy-detailed in Table 3 A brief description of the main globin gene disorders for

which prenatal diagnosis should be offered is given below

HbH disease results from the compound heterozygous state of α0-and

α+-thalassemia ( /-α), or more rarely, from the homozygous state ofnondeletion α+-thalassemia mutations affecting the more dominant α2 gene(αTα/αTα) (22) Individuals with HbH disease have a moderately severe

hypochromic microcytic anemia and produce large amounts of HbH (β4) as aresult of the excess β-chains in the reticulocyte Patients may suffer from

Table 3

Genotype/Phenotype Relationships of the Thalassemias, Sickle-Cell Disease,

and the Various Thalassemia Interactions With Hb Variants

1 Homozygous state

α0-Thalassemia ( / ) Hb Bart’s hydrops fetalis S Blot/PCR

β-Thalassemia

α0-Thalassemia/α+-thalassemia ( /-α) HbH disease S Blot/PCR

α0-Thalassemia/α+-thalassemia ( /αTα) Severe HbH disease S Blot/PCR

Antenatal Diagnosis of Hemoglobinopathies

Mild β+/β0 or severe β+ Variable: intermedia to major PCR

δβ0/β0 or severe β+ Variable: intermedia to major S Blot/PCR

ααα/β0 or severe β+ Mild thalassemia intermedia S Blot/PCR

HbC/β0 or severe β+ Variable: β-thalassemia trait to intermedia PCR

HbE/β0 or severe β+ Variable: intermedia to major PCR

Hb, Hemoglobin; S Blot, Southern blotting; PCR, PCR; HPFH, hereditary persistence of fetal hemoglobin.

42 Oldfatigue, general discomfort, and splenomegaly, but they rarely require hospi-talization and lead relatively normal lives Therefore prenatal diagnosis is notnormally performed for HbH disease There is also a more severe form of HbHdisease, however, arising from the compound heterozygous state of α0-thalas-semia and nondeletion α+-thalassemia ( /αTα) Such patients seem to exhibitmore severe symptoms with a possible requirement of recurrent blood transfu-sions and splenectomy In some situations couples at risk for this more severeform of HbH disease have opted for prenatal diagnosis and termination of an

affected fetus (21).

1.3.2 β-Thalassemia

The β-thalassemias are a heterogeneous group of disorders characterized byeither an absence of β-globin chain synthesis (β0 type) or a severely reducedrate of synthesis (b+ type) (21) The majority of the b0 and b+ type mutationsare called severe mutations because either in the homozygous or compoundheterozygous state they give rise to the phenotype of β-thalassemia major, atransfusion-dependent anemia occurring early in life Some β-thalassemiamutations (the mild β+ type, sometimes designated β++ type) in the homozy-gous state are associated with a milder clinical condition called thalassemiaintermedia Thalassemia intermedia is caused by a wide variety of genotypesincluding mild β-thalassemia genes, δβ-thalassemia, and Hb Lepore Thecoinheritance of α-thalassemia or one of the many determinants resulting in araised HbF level in adult life may cause thalassemia intermedia in patients whoare homozygous for a severe β-thalassemia gene Patients with thalassemiaintermedia present later in life and are capable of maintaining a hemoglobinlevel above 6 g without transfusion In contrast, the phenotype of compoundheterozygotes when one of these mild mutations is inherited with a severemutation is less clear and less predictable This remains a diagnostic and coun-seling problem

1.3.3 Interaction of Thalassemia With Hb Variants

The β-thalassemia mutations and various Hb variants can interact to duce number of thalassemia and sickle-cell disorders for which genetic coun-seling and prenatal diagnosis should be offered These interactions are listed in

pro-Table 3 HbE, HbO Arab, and Hb Lepore interact with β-thalassemia trait toresult in a potentially severe thalassemia disorder Sickle-cell disease is caused

by not only homozygosity for HbS, but also, in varying degrees of severity,from the interaction of HbS with HbC, HbD Punjab, HbO Arab, and β-thalas-semia trait

Antenatal Diagnosis of Hemoglobinopathies 43

2 Materials

1 Kits for DNA extraction as preferred (see Subheading 3.).

2 dNTPs: Add together 50 µL of a 100 mM solution of each dNTP (as purchased) and 3.8 mL of distilled water The 1.25 mM dNTP stock solution should be stored

in frozen aliquots

3 10X Gap-PCR reaction buffer (composition varies according to primers used)

see Subheading 3 and Table 2.

4 Betaine (Sigma-Aldrich Chemical Co Ltd., England)

5 Mineral oil to overlay PCR reactions

6 PCR primers as per Tables 2, 4, 6–8 Dilute aliquots of primer stock solutions to

make a working solution of 1 OD U/mL and store frozen

7 ARMS PCR buffer: 50 mM KCl, 10 mM Tris-HCl, pH 8.3 at room temperature; 1.5 mM MgCl2; 100 mg/mL of gelatin A 10X stock buffer can be prepared by

adding together 0.5 mL of 1 M Tris-HCl, pH 8.3 at room temperature; 1.25 mL of

2 M KCl, 75 µL of 1 M MgCl2; 5 mg of gelatin; and 3.275 mL of distilled water.The stock buffer is heated at 37°C until the gelatin dissolves and then frozen inaliquots

8 Ammonium sulfate buffer for IghJH PCR: 75 mM Tris-HCl, pH 9.0, 20 mM

(NH4)2SO4, 2.0 mM MgCl2, 0.01% Tween, 10% dimethyl sulfoxide (DMSO),

10 mM β-mercaptoethanol (all final concentrations)

9 Taq polymerases and 10X Taq buffers: In my laboratory, AmpliTaq Gold (PE Biosystems) works best for ARMS-PCR/RE digestion assays and Platinum Taq

(Gibco Life Technologies) for gap-PCR (see Subheading 3.).

10 Tris–borate–EDTA (TBE) buffer: 89 mM Tris, 89 mM boric acid,10 mM EDTA,

pH 8.0

11 Agarose

12 Blue running dye:15% Ficoll/0.05% bromophenol blue

13 Ultraviolet (UV) transilluminator and Polaroid land camera

14 0.5 µg/µL Ethidium Bromide

3 Methods

3.1 DNA Extraction

3.1.1 Blood DNA

DNA is normally prepared from blood that is anticoagulated with heparin,

or preferably, EDTA The DNA can be isolated by the standard method ofphenol–chloroform extraction and ethanol precipitation, or by using one of theseveral kits on the market based on salt extraction, protein precipitation, and soforth Sufficient DNA is obtained from 5–10 mL of peripheral blood formolecular analysis and subsequent storage in a DNA bank at –20°C If this isnot required, a much smaller quantity of blood may be used for PCR diagnosis